Identification of a novel HLA-A2-restricted mutated Survivin epitope and induction of specific anti-HCC CTLs that could effectively cross-recognize wild-type Survivin antigen

Abstract

Peptide vaccine based on tumor-associated antigen (TAA), which usually belongs to self-antigen with poor immunogenicity, has been considered as an attractive option for treatment of malignant tumors. The ideal TAA epitopes should have stable affinity to major histocompatibility complex (MHC) molecules and elicit strong anti-tumor immune response. Although point-mutation technology of TAA peptide may increase the binding capability to MHC molecules, some previous studies have revealed that part of the variant peptides results in lymphocyte not to effectively cross-recognize and kill the target tumor expressed wild-type TAA. Here, we designed a novel HLA-A2-restricted mutated TAA Survivin epitope nonapeptide Sur79L2 (KLSSGCAFL) that showed higher binding ability compared to wild-type peptide Sur79 (KHSSGCAFL) in T2-binding assays. To investigate whether Sur79L2 can induce Survivin-specific anti-hepatocellular carcinoma (HCC) response, we stimulated tumor-associated lymphocytes from a HCC patient with Sur79L2 in vitro. IFN-γ release and cytotoxicity assays showed Sur79L2 could effectively cross-recognize and lysis T2 cell plus peptide Sur79 and HCC cell lines (expression of wild-type Survivin antigen) in an HLA-A2-restricted manner. In contrast, peptide Sur95 (ELTLGEFLKL) that has been reported as a very promising anti-tumor epitope in a variety of tumors except HCC were not able to generate detectable cytotoxic immune responses against HCC in this study. Our results suggest that point-mutated peptide Sur79L2 is a new HLA-A2-restricted CTL epitope and may be useful for the immunotherapy for patients with HCC.

Fig. 1

IFN-γ-producing cells were enumerated by ELISPOT assay against peptide Sur95, peptide Sur79 and peptide Sur79L2. Each experiment was performed with 105 cells/well, and the average number of spots was calculated. Negative peptide (NP), HIVpol476, served as a negative control. Concanavalin (CoA) served as a positive control. Bar graphs indicates mean ± SD. *P < 0.05, **P < 0.01 compared to the NP group

Fig. 2

Specific lysis of CTLs generated from different Survivin-derived peptides against peptide-pulsed T2 cells. Immune effector cells were incubated with target cells at the effector/target ratios shown. CTL assays were performed following a 4-h incubation period. E represents peptide-induced CTLs effectors and T represents peptide-pulsed T2 target cells. Cytotoxic T lymphocytes generated from HIVpol476, served as a NP. Data points represent the mean ± SD as measured by quantitation of LDH release. **P < 0.01 compared to the NP group

Fig. 3

FACS analysis for intracellular Granzyme B expression in peptide-induced CD8⁺ CTLs incubated with different target HCC cell lines for 4 h. The flow cytometric plots from the analysis of the response measured in a representative experiment are shown in a. The percentage value in upper right corner of each subpanel indicated the proportion of double positive cells (CD8⁺, GzmB⁺). The responses are measured in triplicate. The percentages of GzmB⁺ CD8⁺ cells for three peptide-induced CTLs against different HCC cell lines are shown in b. Bar graphs indicates mean ± SD. *P < 0.05, **P < 0.01 compared to the No target cell group

Fig. 4

Morphological analysis of the HCC cell lines after incubation with peptide-induced CTLs at 48 h. Cells were visualized under ×200 magnification

Fig. 5

Anti-HLA-A mAb blocked the peptide-specific CTL reaction on target HCC cell lines. HCC target cells were incubated with or without anti-HLA-A2 and anti-HLA-A24 antibody for 1 h at 4 °C. Immune effector cells were incubated with target cells at the effector/target ratios shown. CTL assays were performed following a 4-h incubation period. Data points represent the mean ± SD as measured by quantitation of LDH release. a E: Sur79-specific CTLs, T: HepG2 cells. b E: Sur79L2-specific CTLs, T: HepG2 cells. c E: Sur79L2-specific CTLs, T: Huh-1 cells. *P < 0.05, **P < 0.01 compared to the No blocking group